There is the possibility that protein preparations, particularly plasma fractionation preparations prepared from human plasma as a starting material, may be contaminated with pathogen capable of infecting human, and the problem of viral infection is therefore particularly significant. To date, we have had incidents of viral infection with human immunodeficiency virus (HIV), hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV) and the like, due to transfusion.
These are not incidents in the past. While the frequency of the incidence is strikingly decreasing by exponential numbers after the introduction of a number of techniques, the occurrence of the incident cannot be completely denied even at present.
To prevent propagation of these viruses, various methods are known that inactivate or remove possibly contaminant viruses. For example, a method of heating a protein-containing composition in a liquid state (JP-A-55-145615, JP-A-56-139422, JP-A-56-106594 and the like), a method of heating in a dry state (Japanese Patent Application under PCT laid-open under kohyo No. 58-500548, JP-A-58-213721 and the like), a method comprising contact with trialkylphosphate and/or a surfactant and the like (JP-A-60-51116 and the like), a method comprising UV irradiation (JP-A-7-196531 and the like), a method comprising virus removal membrane (JP-A-9-100239 and the like), and the like are known.
However, every single treatment to inactivate or remove viruses is associated with difficulty in completely inactivating or removing contaminant viruses without losing the protein activity, because the heat treatment leaves viruses highly resistant to heat, the surfactant treatment leaves non-enveloped viruses, and the treatment exclusively by irradiation possibly deactivates protein and the like.
Thus, the above-mentioned methods for inactivating or removing contaminant viruses are currently used in suitable combinations for this purpose.
Fibrinogen is obtained from a fraction first precipitated during the process of alcohol plasma fractionation. Therefore, it is susceptible to the consequence of contamination once a virus invades plasma. Parvovirus B19 (hereinafter B19) is particularly drawing much attention lately. This virus does not have an envelope, is resistant to heat and has an extremely small single-particle size of about 18-25 nm, due to which properties its inactivation/removal is desired and is being investigated particularly in the field of plasma fractionation preparation.
A method using a virus removal membrane is naturally capable of removing viruses having a greater molecule size than its pore size upon filtration, but otherwise when the virus is smaller in size than the pore size. In addition, the use of a removal membrane having too small a pore size results in clogging with a sample and the like, which makes filtration itself difficult. Furthermore, lower flow rates in parallel with the sample amounts during a membrane treatment give rise to many problems such as limited sample amount to be treated and a longer treatment time.
Particularly, when a currently commercially available porous membrane having a membrane pore size of less than 35 nm was used for purification of fibrinogen, the above-mentioned problems of clogging, limited sample amount to be treated and longer treatment time actually occurred due to the molecular weight of fibrinogen and the like. Consequently, only a porous membrane having a membrane pore size of not less than 35 nm can be used. As mentioned above, Parvovirus (B19) has a single-particle size of about 18-25. nm Accordingly, the virus cannot be removed by a sole use of PLANOVA 35 N or PLANOVA 75N (both trademarks, now commercially available porous membrane having a pore size of 35 nm or 75 nm, manufactured by Asahi Kasei Corporation), or even by a method using these virus removal membranes in multi-steps.
Therefore, there has been a demand for a method capable of providing a less virus-infectious and safe protein preparation by a convenient treatment method.